Formation of persistent free radicals from epigallocatechin Gallate in tea processing and their implications on DNA damage and cell cytotoxicity.

Tea is widely consumed in both beverages and food. Epigallocatechin gallate (EGCG) is the most crucial active ingredient in tea. Currently, knowledges on transformation processes of EGCG during tea processing are lacking. Understanding the chemical reactions of EGCG and its products during tea processing is important for assessing the safety of tea-containing food. Here, we revealed the formation of persistent free radicals (PFRs) from EGCG under the influence of heating and light irradiation, which was substantiated with evidence. These PFRs exhibited stability for >30 min in simulated gastric fluid. Furthermore, we observed potential effects of these PFRs on DNA damage and cell cytotoxicity in vitro. By combining electron paramagnetic resonance spectrometer with Fourier transform ion cyclotron resonance mass spectrometry, we elucidated the pathways involved in free radical formation. These findings are expected to contribute to a comprehensive understanding of free radical chemistry in tea-containing food.

A drug repurposing study identifies novel FOXM1 inhibitors with in vitro activity against breast cancer cells.

FOXM1, a proto-oncogenic transcription factor, plays a critical role in cancer development and treatment resistance in cancers, particularly in breast cancer. Thus, this study aimed to identify potential FOXM1 inhibitors through computational screening of drug databases, followed by in vitro validation of their inhibitory activity against breast cancer cells. In silico studies involved pharmacophore modeling using the FOXM1 inhibitor, FDI-6, followed by virtual screening of DrugBank and Selleckchem databases. The selected drugs were prepared for molecular docking, and the crystal structure of FOXM1 was pre-processed for docking simulations. In vitro studies included MTT assays to assess cytotoxicity, and Western blot analysis to evaluate protein expression levels. Our study identified Pantoprazole and Rabeprazole as potential FOXM1 inhibitors through in silico screening and molecular docking. Molecular dynamics simulations confirmed stable interactions of these drugs with FOXM1. In vitro experiments showed both Pantoprazole and Rabeprazole exhibited strong FOXM1 inhibition at effective concentrations and that showed inhibition of cell proliferation. Rabeprazole showed the inhibitor activity at 10 µM in BT-20 and MCF-7 cell lines. Pantoprazole exhibited FOXM1 inhibition at 30 µM and in BT-20 cells and at 70 µM in MCF-7 cells, respectively. Our current study provides the first evidence that Rabeprazole and Pantoprazole can bind to FOXM1 and inhibit its activity and downstream signaling, including eEF2K and pEF2, in breast cancer cells. These findings indicate that rabeprazole and pantoprazole inhibit FOXM1 and breast cancer cell proliferation, and they can be used for FOXM1-targeted therapy in breast or other cancers driven by FOXM1.

Exploring the Phytochemical Composition and the Bioactive Properties of Malbec and Torrontés Wine Pomaces from the Calchaquíes Valleys (Argentina) for Their Sustainable Exploitation.

Hydroalcoholic extracts from Malbec and Torrontés wine pomaces (Vitis vinifera L.) originating from the high-altitude vineyards of Argentina's Calchaquí Valleys were characterized. Total phenolics, hydroxycinnamic acids, orthodiphenols, anthocyanins, non-flavonoid phenolics, total flavonoids, flavones/flavonols, flavanones/dihydroflavonols, and tannins were quantified through spectrophotometric methods, with the Malbec extract exhibiting higher concentrations in most of phytochemical groups when compared to Torrontés. HPLC-DAD identified more than 30 phenolic compounds in both extracts. Malbec displayed superior antiradical activity (ABTS cation, nitric oxide, and superoxide anion radicals), reduction power (iron, copper, and phosphomolybdenum), hypochlorite scavenging, and iron chelating ability compared to Torrontés. The cytotoxicity assessments revealed that Torrontés affected the viability of HT29-MTX and Caco-2 colon cancer cells by 70% and 50%, respectively, at the highest tested concentration (1 mg/mL). At the same time, both extracts did not demonstrate acute toxicity in Artemia salina or in red blood cell assays at 500 µg/mL. Both extracts inhibited the lipoxygenase enzyme (IC50: 154.7 and 784.7 µg/mL for Malbec and Torrontés), with Malbec also reducing the tyrosinase activity (IC50: 89.9 µg/mL), and neither inhibited the xanthine oxidase. The substantial phenolic content and diverse biological activities in the Calchaquí Valleys' pomaces underline their potentialities to be valorized for pharmaceutical, cosmetic, and food industries.

Insights from HIV-1 vaccine and passive immunization efficacy trials.

An effective HIV-1 vaccine is still not available, and most vaccine efficacy trials conducted over the years resulted in no significant overall protection. Here we highlight several insights gained from these trials as well as emerging questions that may be important for further guidance to advance current research directions.

Human cytomegalovirus UL23 exploits PD-L1 inhibitory signaling pathway to evade T cell-mediated cytotoxicity.

Human cytomegalovirus (HCMV), a widely prevalent human beta-herpesvirus, establishes lifelong persistence in the host following primary infection. In healthy individuals, the virus is effectively controlled by HCMV-specific T cells and typically exhibits asymptomatic. The T cell immune response plays a pivotal role in combating HCMV infection, while HCMV employs various strategies to counteract it within the host. Previously, we reported that UL23, a tegument protein of HCMV, facilitates viral immune evasion from interferon-gamma (IFN-γ) responses, and it is well known that IFN-γ is mainly derived from T cells. However, the involvement of UL23 in viral immune evasion from T cell-mediated immunity remains unclear. Herein, we present compelling evidence that UL23 significantly enhances viral resistance against T cell-mediated cytotoxicity during HCMV infection from the co-culture assays of HCMV-infected cells with T cells. We found that IFN-γ plays a major role in regulating T cell cytotoxicity mediated by UL23. More interestingly, we demonstrated that UL23 not only regulates the IFN-γ downstream responses but also modulates the IFN-γ secretion by regulating T cell activities. Further experiments indicate that UL23 upregulates the expression and signaling of programmed death ligand 1 (PD-L1), which is responsible for inhibiting multiple aspects of T cell activities, including activation, apoptosis, and IFN-γ secretion, as determined through RNA-seq analysis and inhibitor-blocking experiments, ultimately facilitating viral replication and spread. Our findings highlight the potential role of UL23 as an alternative antagonist in suppressing T cell cytotoxicity and unveil a novel strategy for HCMV to evade T cell immunity. IMPORTANCE: T cell immunity is pivotal in controlling primary human cytomegalovirus (HCMV) infection, restricting periodic reactivation, and preventing HCMV-associated diseases. Despite inducing a robust T cell immune response, HCMV has developed sophisticated immune evasion mechanisms that specifically target T cell responses. Although numerous studies have been conducted on HCMV-specific T cells, the primary focus has been on the impact of HCMV on T cell recognition via major histocompatibility complex molecules. Our studies show for the first time that HCMV exploits the programmed death ligand 1 (PD-L1) inhibitory signaling pathway to evade T cell immunity by modulating the activities of T cells and thereby blocking the secretion of IFN-γ, which is directly mediated by HCMV-encoded tegument protein UL23. While PD-L1 has been extensively studied in the context of tumors and viruses, its involvement in HCMV infection and viral immune evasion is rarely reported. We observed an upregulation of PD-L1 in normal cells during HCMV infection and provided strong evidence supporting its critical role in UL23-induced inhibition of T cell-mediated cytotoxicity. The novel strategy employed by HCMV to manipulate the inhibitory signaling pathway of T cell immune activation for viral evasion through its encoded protein offers valuable insights for the understanding of HCMV-mediated T cell immunomodulation and developing innovative antiviral treatment strategies.

Human serum albumin directed formation of cadmium telluride quantum dots: Applications in biosensing, anti-bacterial activities and cell cytotoxicity measurements.

Although cadmium-based quantum dots (QDs) are highly promising candidates for numerous biological applications, their intrinsic toxicity limits their pertinency in living systems. Surface functionalization of QDs with appropriate molecules could reduce the toxicity level. Herein, we have synthesized the smaller sized (1-5 nm) aqueous-compatible biogenic CdTe QDs using human serum albumin (HSA) as a surface passivating agent via a greener approach. HSA-functionalized CdTe QDs have been explored in multiple in vitro sensing and biological applications, namely, (1) sensing, (2) anti-bacterial and (3) anti-cancer properties. Using CdTe-HSA QDs as a fluorescence probe, a simple fluorometric method has been developed for highly sensitive and selective detection of blood marker bilirubin and hazardous Hg2+ ion with a limit of detection (LOD) of 3.38 and 0.53 ng/mL, respectively. CdTe-HSA QDs also acts as a sensor for standard antibiotics, tetracycline and rifampicin with LOD values of 41.34 and 114.99 ng/mL, respectively. Nano-sized biogenic CdTe-HSA QDs have shown promising anti-bacterial activities against both gram-negative, E. coli and gram-positive, E. faecalis strains confirming more effectiveness against E. faecalis strains. The treatment of human cervical cancer cell lines (HeLa cells) with the synthesized QDs reflected the proficient cytotoxic properties of QDs.

Lacticaseibacillus casei K11 exerts immunomodulatory effects by enhancing natural killer cell cytotoxicity via the extracellular regulated-protein kinase pathway.

PURPOSE: Probiotics can serve as immunomodulators that regulate the activation of immune cells. This study aimed to screen potential probiotic strains that can enhance NK cell toxicity to improve host immunity. METHODS: In this investigation, we examined three potential probiotic strains, namely Lactiplantibacillus plantarum YZX21 (YZX21), Bifidobacterium bifidum FL-276.1 (FL-276.1) and Lacticaseibacillus casei K11 (K11), to assess their capacity in modulating NK cytotoxicity both in vitro and in vivo, while elucidating the underlying mechanisms involved. RESULTS: The findings demonstrated that K11 exhibited superior efficacy in enhancing NK cytotoxicity. Subsequent analysis revealed that K11 significantly augmented the secretion of perforin and granzyme B by NK cells through activation of receptors NKp30 and NKp46 via the extracellular signal-regulated kinase (ERK) pathway. Furthermore, heat-inactivated K11 also enhanced NK cell activity to an extent comparable to live bacteria, with lipoteichoic acid from K11 identified as a crucial factor mediating the activation of NK cell cytotoxicity. CONCLUSION: Our study suggests that K11 may have potential applications as probiotics or postbiotics for regulating NK cell cytotoxicity to enhance immunity.

Soluble MICA in endometriosis pathophysiology: Impairs NK cell degranulation and effector functions.

PROBLEM: Endometriosis exhibits several immune dysfunctions, including deficient natural killer (NK) cell cytotoxicity. MICA (MHC class I chain-related molecule A) is induced by biological stress and soluble MICA (sMICA) negatively modulates the expression of the activating receptor, NKG2D, reducing NK cells activities. We investigated the involvement of soluble MICA in NK cell-deficient activity in endometriosis. METHODS OF STUDY: sMICA levels (serum and peritoneal fluid-PF) were evaluated by ELISA. Circulating NK cell subsets quantification and its NKG2D receptor expression, NK cell cytotoxicity and CD107a, IFN-γ and IL-10 expressions by NK cells stimulated with K562 cells were determined by flow cytometry. RESULTS: We found higher sMICA levels (serum and PF) in endometriosis, especially in advanced and deep endometriosis. Endometriosis presented lower percentages of CD56dim CD16+ cytotoxic cells and impaired NK cell responses upon stimulation, resulting in lower CD107a and IFN-γ expressions, and deficient NK cell cytotoxicity. NK cell stimulation in the MICA-blocked condition (mimicking the effect of sMICA) showed decreased cytotoxicity in initial endometriosis stages and the emergence of a negative correlation between CD107a expression and sMICA levels. CONCLUSIONS: We suggest that soluble MICA is a potential player in endometriosis pathophysiology with involvement in disease progression and severity, contributing to NK cell impaired IFN-γ response and degranulation. NK cell compartment exhibits multiple perturbations, including quantitative deficiency and impaired cytotoxicity, contributing to inadequate elimination of ectopic endometrial tissue.

Antibody dependent cellular cytotoxicity-inducing anti-EGFR antibodies as effective therapeutic option for cutaneous melanoma resistant to BRAF inhibitors.

Introduction: About 50% of cutaneous melanoma (CM) patients present activating BRAF mutations that can be effectively targeted by BRAF inhibitors (BRAFi). However, 20% of CM patients exhibit intrinsic drug resistance to BRAFi, while most of the others develop adaptive resistance over time. The mechanisms involved in BRAFi resistance are disparate and globally seem to rewire the cellular signaling profile by up-regulating different receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). RTKs inhibitors have not clearly demonstrated anti-tumor activity in BRAFi resistant models. To overcome this issue, we wondered whether the shared up-regulated RTK phenotype associated with BRAFi resistance could be exploited by using immune weapons as the antibody-dependent cell cytotoxicity (ADCC)-mediated effect of anti-RTKs antibodies, and kill tumor cells independently from the mechanistic roots. Methods and results: By using an in vitro model of BRAFi resistance, we detected increased membrane expression of EGFR, both at mRNA and protein level in 4 out of 9 BRAFi-resistant (VR) CM cultures as compared to their parental sensitive cells. Increased EGFR phosphorylation and AKT activation were observed in the VR CM cultures. EGFR signaling appeared dispensable for maintaining resistance, since small molecule-, antibody- and CRISPR-targeting of EGFR did not restore sensitivity of VR cells to BRAFi. Importantly, immune-targeting of EGFR by the anti-EGFR antibody cetuximab efficiently and specifically killed EGFR-expressing VR CM cells, both in vitro and in humanized mouse models in vivo, triggering ADCC by healthy donors' and patients' peripheral blood cells. Conclusion: Our data demonstrate the efficacy of immune targeting of RTKs expressed by CM relapsing on BRAFi, providing the proof-of-concept supporting the assessment of anti-RTK antibodies in combination therapies in this setting. This strategy might be expected to concomitantly trigger the crosstalk of adaptive immune response leading to a complementing T cell immune rejection of tumors.

CAR-mediated targeting of NK cells overcomes tumor immune escape caused by ICAM-1 downregulation.

BACKGROUND: The antitumor activity of natural killer (NK) cells can be enhanced by specific targeting with therapeutic antibodies that trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or by genetic engineering to express chimeric antigen receptors (CARs). Despite antibody or CAR targeting, some tumors remain resistant towards NK cell attack. While the importance of ICAM-1/LFA-1 interaction for natural cytotoxicity of NK cells is known, its impact on ADCC induced by the ErbB2 (HER2)-specific antibody trastuzumab and ErbB2-CAR-mediated NK cell cytotoxicity against breast cancer cells has not been investigated. METHODS: Here we used NK-92 cells expressing high-affinity Fc receptor FcγRIIIa in combination with trastuzumab or ErbB2-CAR engineered NK-92 cells (NK-92/5.28.z) as well as primary human NK cells combined with trastuzumab or modified with the ErbB2-CAR and tested cytotoxicity against cancer cells varying in ICAM-1 expression or alternatively blocked LFA-1 on NK cells. Furthermore, we specifically stimulated Fc receptor, CAR and/or LFA-1 to study their crosstalk at the immunological synapse and their contribution to degranulation and intracellular signaling in antibody-targeted or CAR-targeted NK cells. RESULTS: Blockade of LFA-1 or absence of ICAM-1 significantly reduced cell killing and cytokine release during trastuzumab-mediated ADCC against ErbB2-positive breast cancer cells, but not so in CAR-targeted NK cells. Pretreatment with 5-aza-2'-deoxycytidine induced ICAM-1 upregulation and reversed NK cell resistance in ADCC. Trastuzumab alone did not sufficiently activate NK cells and required additional LFA-1 co-stimulation, while activation of the ErbB2-CAR in CAR-NK cells induced efficient degranulation independent of LFA-1. Total internal reflection fluorescence single molecule imaging revealed that CAR-NK cells formed an irregular immunological synapse with tumor cells that excluded ICAM-1, while trastuzumab formed typical peripheral supramolecular activation cluster (pSMAC) structures. Mechanistically, the absence of ICAM-1 did not affect cell-cell adhesion during ADCC, but rather resulted in decreased signaling via Pyk2 and ERK1/2, which was intrinsically provided by CAR-mediated targeting. Furthermore, while stimulation of the inhibitory NK cell checkpoint molecule NKG2A markedly reduced FcγRIIIa/LFA-1-mediated degranulation, retargeting by CAR was only marginally affected. CONCLUSIONS: Downregulation of ICAM-1 on breast cancer cells is a critical escape mechanism from trastuzumab-triggered ADCC. In contrast, CAR-NK cells are able to overcome cancer cell resistance caused by ICAM-1 reduction, highlighting the potential of CAR-NK cells in cancer immunotherapy.

Clostridioides difficile Infection: Diagnosis and Treatment Challenges.

Clostridioides difficile is the most important cause of healthcare-associated diarrhea in the United States. The high incidence and recurrence rates of C. difficile infection (CDI), associated with high morbidity and mortality, pose a public health challenge. Although antibiotics targeting C. difficile bacteria are the first treatment choice, antibiotics also disrupt the indigenous gut flora and, therefore, create an environment that is favorable for recurrent CDI. The challenge of treating CDI is further exacerbated by the rise of antibiotic-resistant strains of C. difficile, placing it among the top five most urgent antibiotic resistance threats in the USA. The evolution of antibiotic resistance in C. difficile involves the acquisition of new resistance mechanisms, which can be shared among various bacterial species and different C. difficile strains within clinical and community settings. This review provides a summary of commonly used diagnostic tests and antibiotic treatment strategies for CDI. In addition, it discusses antibiotic treatment and its resistance mechanisms. This review aims to enhance our current understanding and pinpoint knowledge gaps in antimicrobial resistance mechanisms in C. difficile, with an emphasis on CDI therapies.

Anticarcinogenic Effects of Gold Nanoparticles and Metformin Against MCF-7 and A549 Cells.

Metformin is commonly prescribed to people with diabetes. Metformin has been shown in previous studies to be able to prevent the growth of cancer cells. This study aims to investigate the effects of metformin and gold nanoparticles in MCF7 breast cancer and A549 lung cell lines. The effects of metformin and gold nanoparticles on MCF7 breast cancer and A549 lung cells were determined on cells grown in 24 h cell culture. MCF-7 and A549 cells were incubated for 24 h with the treatment of escalating molar concentrations of ifosfamide. The MTT assay was used to determine the cytotoxicity of metformin toward MCF7 and A549 cell lines. The expression of Bax, BCL2, PI3K, Akt3, mTOR, Hsp60, Hsp70, and TNF-α was measured by RT-PCR. Metformin and gold nanoparticles inhibited the proliferation of MCF-7 and A549 cells in a dose and time-dependent manner with an IC50 value of 5 µM and 10 µg/mL. RT-PCR assays showed ifosfamide + metformin + gold nanoparticles significantly reduced the expression of BCL2, PI3K, Akt3, mTOR, Hsp60 and Hsp70 and increased the expression of TNF-α and Bax. The findings obtained in this study suggest that further studies should be conducted, and metformin and gold nanoparticles can be used in breast cancer and lung cancer treatments.

RIPK3 promotes islet amyloid-induced ß-cell loss and glucose intolerance in a humanized mouse model of type 2 diabetes.

OBJECTIVE: Aggregation of human islet amyloid polypeptide (hIAPP), a ß-cell secretory product, leads to islet amyloid deposition, islet inflammation and ß-cell loss in type 2 diabetes (T2D), but the mechanisms that underlie this process are incompletely understood. Receptor interacting protein kinase 3 (RIPK3) is a pro-death signaling molecule that has recently been implicated in amyloid-associated brain pathology and ß-cell cytotoxicity. Here, we evaluated the role of RIPK3 in amyloid-induced ß-cell loss using a humanized mouse model of T2D that expresses hIAPP and is prone to islet amyloid formation. METHODS: We quantified amyloid deposition, cell death and caspase 3/7 activity in islets isolated from WT, Ripk3-/-, hIAPP and hIAPP; Ripk3-/- mice in real time, and evaluated hIAPP-stimulated inflammation in WT and Ripk3-/- bone marrow derived macrophages (BMDMs) in vitro. We also characterized the role of RIPK3 in glucose stimulated insulin secretion (GSIS) in vitro and in vivo. Finally, we examined the role of RIPK3 in high fat diet (HFD)-induced islet amyloid deposition, ß-cell loss and glucose homeostasis in vivo. RESULTS: We found that amyloid-prone hIAPP mouse islets exhibited increased cell death and caspase 3/7 activity compared to amyloid-free WT islets in vitro, and this was associated with increased RIPK3 expression. hIAPP; Ripk3-/- islets were protected from amyloid-induced cell death compared to hIAPP islets in vitro, although amyloid deposition and caspase 3/7 activity were not different between genotypes. We observed that macrophages are a source of Ripk3 expression in isolated islets, and that Ripk3-/- BMDMs were protected from hIAPP-stimulated inflammatory gene expression (Tnf, Il1b, Nos2). Following 52 weeks of HFD feeding, islet amyloid-prone hIAPP mice exhibited impaired glucose tolerance and decreased ß-cell area compared to WT mice in vivo, whereas hIAPP; Ripk3-/- mice were protected from these impairments. CONCLUSIONS: In conclusion, loss of RIPK3 protects from amyloid-induced inflammation and islet cell death in vitro and amyloid-induced ß-cell loss and glucose intolerance in vivo. We propose that therapies targeting RIPK3 may reduce islet inflammation and ß-cell loss and improve glucose homeostasis in the pathogenesis of T2D.

Immunohistochemical expression of perforin in adult systemic lupus erythematosus associated macrophage activation syndrome: Clinicohematological correlation and literature review.

OBJECTIVE: To study the bone marrow (BM) immunohistomorphological characteristics in adult systemic lupus erythematosus (SLE) associated macrophage activation syndrome (SLE-MAS). MATERIALS AND METHODS: Immunohistochemical (IHC) expression of CD3, CD8, perforin (PFN), and CD163 was studied on BM trephine biopsies from 30 cytopenic adult SLE cases (male: female = 1:5, age; 24 years, range; 19-32) and compared them with ten age matched controls. Clinicopathological parameters were compared among the cases likely (L) or unlikely (U) to have MAS using probability scoring criteria. The best cut off laboratory parameters to discriminate between the two were obtained through receiver operator curve (ROC) analysis. RESULTS: MAS occurred in 12/30 (40%) cases and was more commonly associated with prior immunosuppressive therapy (p = .07), ≥ 3 system involvement (p = .09), lower fibrinogen (p < .01), increased triglyceride (p = .002), increased BM hemophagocytosis (p = .002), and higher MAS score [185 (176-203) vs. 105 (77-119), p < .01] than MAS-U subgroup. Although PFN+CD8+ T lymphocytes significantly decreased among cases than controls (p < .05), it was comparable between MAS-L and MAS-U subgroups. Fibrinogen (< 2.4 g/L, AUC; 0.93, p < .01), hemophagocytosis score (> 1.5, AUC; 0.71, p = .03), and an MAS probability score of ≥ 164 (AUC; 1, p < .01) discriminated MAS from those without MAS. CONCLUSION: We noted a decrease in perforin mediated CD8 + T cell cytotoxicity in SLE. Immunohistochemical demonstration of the same along with histiocytic hemophagocytosis on BM biopsy may be useful adjunct in early diagnosis and management of MAS in SLE.

Therapeutic applications of sustainable new chitosan derivatives and its nanocomposites: Fabrication and characterization.

Chitosan (CS) is a biologically active biopolymer used in different medical applications due to its biodegradability, biocompatibility, and nontoxicity. Nanotechnology is an exciting and quick developing field in medical applications. Nanoparticles have shown great potential in the treatment of cancer and inflammation. In the present work modification of chitosan and its (Ag, Au, or ZnO) nanocomposites by N-aminophthalimide (NAP) occurred through the reaction with epichlorohydrin (ECH) as a crosslinker in the presence or absence of glutaraldehyde (GA) under different reaction conditions using microwave irradiation to give modified chitosan derivatives CS-2, CS-6, and their nanocomposites. Modified chitosan derivatives were characterized using different tools. CS-2 and CS-6 derivatives displayed enhancement of thermal stability and crystallinity compared to chitosan. Additionally, CS-2, CS-6, and their nanocomposites exhibited improvements in antitumor activity against HeLa cancer cells and enzymatic inhibitory against trypsin and α-chymotrypsin enzymes compared to chitosan. However, CS-2 revealed the highest cell growth inhibition% toward HeLa cells (89.02 ± 1.46 %) and the enzymatic inhibitory toward α-chymotrypsin enzyme (17.13 ± 1.59 %). Furthermore, CS-Au-2 showed the highest enzymatic inhibitory against trypsin enzyme (28.14 ± 1.76 %). These results suggested that the new chitosan derivatives CS-2, CS-6, and their nanocomposites could be a platform for medical applications against HeLa cells, trypsin, and α-chymotrypsin enzymes.

Impact of Wooden Breast myopathy on in vitro protein digestibility, metabolomic profile, and cell cytotoxicity of cooked chicken breast meat.

This study investigated the impacts of Wooden Breast (WB) abnormality on in vitro protein digestibility and cytotoxicity of cooked chicken breast meat. Chicken breasts without (non-WB, n = 6) or with severe WB condition (WB, n = 6) were cooked and subjected to static in vitro protein digestion. The results showed no significant differences in free-NH2, degree of hydrolysis and distribution of peptide molecular weight between non-WB and WB samples at late intestinal digestion (P5), suggesting no adverse effects of WB on protein digestibility. Based on peptidomic analysis, P5 fraction of WB showed greater content of peptides with oxidative modification than that of non-WB. Untargeted metabolomics did not find any metabolites with potential toxicity either in non-WB and WB. Hydrolyzed non-WB and WB (1.56-100 µg/mL) did not affect viability of Caco-2 and Vero cells but addition of WB samples reduced Caco-2 cell viability compared with non-WB.

Scaling-up of carbon dots hydrothermal synthesis from sugars in a continuous flow microreactor system for biomedical application as in vitro antimicrobial drug nanocarrier.

Carbon dots (CDs) are a new class of nanomaterials exhibiting high biocompatibility, water solubility, functionality, and tunable fluorescence (FL) property. Due to the limitations of batch hydrothermal synthesis in terms of low CDs yield and long synthesis duration, this work aimed to increase its production capacity through a continuous flow reactor system. The influence of temperature and time was first studied in a batch reactor for glucose, xylose, sucrose and table sugar precursors. CDs synthesized from sucrose precursor exhibited the highest quantum yield (QY) (175.48%) and the average diameter less than 10 nm (~6.8 ± 1.1 nm) when synthesized at 220°C for 9 h. For a flow reactor system, the best condition for CDs production from sucrose was 1 mL min-1 flow rate at 280°C, and 0.2 MPa pressure yielding 53.03% QY and ~ 6.5 ± 0.6 nm average diameter (6.6 mg min-1 of CDs productivity). CDs were successfully used as ciprofloxacin (CP) nanocarrier for antimicrobial activity study. The cytotoxicity study showed that no effect of CDs on viability of L-929 fibroblast cells was detected until 1000 µg mL-1 CDs concentration. This finding demonstrates that CDs synthesized via a flow reactor system have a high zeta potential and suitable surface properties for nano-theranostic applications.

The loss of B7-H4 expression in breast cancer cells escaping from T cell cytotoxicity contributes to epithelial-to-mesenchymal transition.

BACKGROUND: B7 homology 4 (B7-H4), a potential target for cancer therapy, has been demonstrated to inhibit T cell cytotoxicity in the early stages of breast cancer. However, B7-H4 manipulating breast tumor immune microenvironment (TIME) in the tumor progression remains unknown. METHODS: We engineered T cells with B7-H4-specific chimeric antigen receptors (CARs) and performed a T cell co-culture assay to characterize B7-H4 expression level in breast cancer cells escaping from T cell cytotoxicity. We generated B7-H4 knockout (KO) and overexpression (OE) breast cancer cells to determine the epithelial-to-mesenchymal transition (EMT) and stemness characteristics in vitro and in vivo, including tumor proliferation, migration, metastasis and chemoresistance. The Cancer Genome Atlas breast cancer database was accessed to investigate the correlation between B7-H4 expression levels and EMT characteristics in patients with breast cancer. RESULTS: Our result found that B7-H4 expression level was significantly reduced in a subset of breast cancer cells that escaped from the cytotoxicity of B7-H4 CAR-T cells. Compared with wild type cells, B7-H4 KO cells prompt EMT and stemness characteristics, including migration, invasion and metastasis, and OE cells vice versa. The increase in H3K27me3 in KO cells confirmed the epigenetic reprogramming of cancer stem cells. The IC50 of doxorubicin or oxaliplatin significantly increased in KO cells, which was in agreement with a decrease in OE cells. Moreover, a trend of downregulated B7-H4 from stage I to stage II breast cancer patients indicates that the low-expressing B7-H4 breast cancer cells escaping from TIME have spread to nearby breast lymph nodes in the cancer progression. CONCLUSIONS: Our study illuminates the novel role of renouncing B7-H4 in breast cancer cells through immune escape, which contributes to EMT processes and provides new insights for breast cancer treatments.

Endogenous H3.3K27M derived peptide restricted to HLA-A∗02:01 is insufficient for immune-targeting in diffuse midline glioma.

Diffuse midline glioma (DMG) is a childhood brain tumor with an extremely poor prognosis. Chimeric antigen receptor (CAR) T cell therapy has recently demonstrated some success in DMG, but there may a need to target multiple tumor-specific targets to avoid antigen escape. We developed a second-generation CAR targeting an HLA-A∗02:01 restricted histone 3K27M epitope in DMG, the target of previous peptide vaccination and T cell receptor-mimics. These CAR T cells demonstrated specific, titratable, binding to cells pulsed with the H3.3K27M peptide. However, we were unable to observe scFv binding, CAR T cell activation, or cytotoxic function against H3.3K27M+ patient-derived models. Despite using sensitive immunopeptidomics, we could not detect the H3.3K27M26-35-HLA-A∗02:01 peptide on these patient-derived models. Interestingly, other non-mutated peptides from DMG were detected bound to HLA-A∗02:01 and other class I molecules, including a novel HLA-A3-restricted peptide encompassing the K27M mutation and overlapping with the H3 K27M26-35-HLA-A∗02:01 peptide. These results suggest that targeting the H3 K27M26-35 mutation in context of HLA-A∗02:01 may not be a feasible immunotherapy strategy because of its lack of presentation. These findings should inform future investigations and clinical trials in DMG.

A Genetically Encoded Dark-to-Bright Biosensor for Visualisation of Granzyme-Mediated Cytotoxicity.

Granzyme B (GZMB) is a key enzyme released by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells to induce apoptosis in target cells. We designed a novel fluorogenic biosensor which is able to assess GZMB activity in a specific and sensitive manner. This cleavage-responsive sensor for T cell activity level (CRSTAL) is based on a fluorescent protein that is only activated upon cleavage by GZMB or caspase-8. CRSTAL was tested in stable cell lines and demonstrated a strong and long-lasting fluorescence signal upon induction with GZMB. It can detect GZMB activity not only by overexpression of GZMB in target cells but also following transfer of GZMB and perforin from effector cells during cytotoxicity. This feature has significant implications for cancer immunotherapy, particularly in monitoring the efficacy of chimeric antigen receptor (CAR)-T cells. CAR-T cells are a promising therapy option for various cancer types, but monitoring their activity in vivo is challenging. The development of biosensors like CRSTAL provides a valuable tool for monitoring of CAR-T cell activity. In summary, CRSTAL is a highly sensitive biosensor that can detect GZMB activity in target cells, providing a means for evaluating the cytotoxic activity of immune cells and monitoring T cell activity in real time.